Tripeptides and derivatives thereof for cosmetic application in order to improve skin structure

ABSTRACT

The invention relates to compounds and to the cosmetically acceptable salts thereof, which correspond to general formula (I), 
     
       
         
         
             
             
         
       
     
     wherein: R 1  represents H, —C(O)—R 6 , —SO 2 —R 6 or —C(O)—XR 6 ; R 2  and R 4 , independent of one another, represent (CH 2 ) n —NH 2  or (CH 2 ) 3 —NHC(NH)NH 2 ; n equals 1 4; R 3  represents linear or branched C 1 -C 4  alkyl that is optionally substituted by hydroxy; R 5  and R 6 , independent of one another, represent hydrogen, optionally substituted (C 1 -C 24 )alkyl, optionally substituted C 2 -C 24  alkenyl, optionally substituted phenyl, optionally substituted phenyl-C 1 -C 4  alkyl or 9-fluorenyl-methyl; X represents oxygen (—O—) or NH—; or XR 5  with X═O also represents the esters of a-tocopherol, tocotrienol or retinol, with the provision that R 1  and R 5  do not represent hydrogen and X does not represent oxygen at the same time. The invention also relates to the production of the compounds of general formula (I) and to a cosmetically active composition that contains at least one compound of formula (I).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/555,994, filed on Nov. 8, 2005, which U.S. application Ser. No.10/555,994 is the National Stage of International Application No.PCT/CH2004/000278, filed May 7, 2004, which claims the benefit of CH807/03, filed May 8, 2003, the entire contents of all of which areincorporated by reference herein.

BACKGROUND OF THE INVENTION

It is known that endogenous (age-related) or exogenous (light-induced)aging leads to an irreversible degeneration of tissues, in particular ofskin. These modifications result from a reduction of anabolic reactions(syntheses) and an increase of catabolic reactions (degradation) ofcollagen and elastin, the two main constituents of the skin matrix.

The synthesis reactions in the skin matrix are mostly regulated bypolypeptides, so-called growth factors and cytokines. Among thesepeptides, TGFβ1 is one of the most important regulators involved in thesynthesis reactions of this skin matrix. It is secreted in the matrix bykeratinocytes and fibroblasts in a latent form and has to be activatedin order to be recognized by the cell receptors and to be able to inducea biological response (collagen and elastin synthesis). Two forms oflatent TGFβ1 are available:

-   -   a small, latent complex composed of 2 TGFβ-chains that are        non-covalently bound to a so-called “latency associated protein”        (LAP).    -   a large, latent TGFβ1-complex, in which the small, latent        TGFβ1-complex is covalently bound (disulfide bonds) by the LAP        to another, so-called “latent TGFβ binding protein” (LTBP). It        has been found recently that in human skin this large, latent        TGFβ1-complex is associated to fibrillin, a microfibril-forming        molecule; these microfibrils are themselves bound to elastin.        Thus, the large, latent TGFβ1-complex constitutes the greatest        reservoir of latent TGFβ1 in skin.

There are several physiological mechanisms to activate TGFβ1. The mainmethod in vivo is the activation of latent TGFβ1 by thrombospondin-1(TSP-1), a protein secreted by the skin cells. This activation bases onthe interaction between the LAP of the latent TGFβ1 and the tripeptidesequence RFK (Arg-Phe-Lys) of TSP-1, XFX (with X=basic amino acid) beingthe smallest sequence required for the activation of latent TGFβ1.

During the aging process the bioavailability and the TGFβ1 activity arereduced by a decreased genetic expression and a modified capacity offixing to fibroblast receptors. These modifications cause weakenedsynthesis reactions of the elastin and collagen fibres. The degradationreactions in the skin matrix are mainly produced by proteolytic enzymes,the matrix proteinases (MMPs).

MMP-1 (or collagenase) and MMP-2 (or gelatinase A) secreted by skinfibroblasts are involved in the chrono-induced aging process. Theirnumber increases in aging skin, leading to a modification of collagenand elastin fibres. In the photo-induced aging process the MMP-9 (orgelatinase B) and MMP-3 (leukocyte elastase) are involved. They aresecreted by keratinocytes and/or polynuclear neutrophils duringUV-induced, inflammatory processes, whereby the elastin and collagenfibres are degraded and reduced (elastose).

Consequently, the decreased anabolism and the increased catabolism ofthe macromolecules in the skin matrix lead to an imbalance that isresponsible for the appearing of the following clinical symptoms: skinatrophy, loss of the mechanical properties with relief and elasticityloss, skin flabbiness, deep mimic wrinkles, accelerated formation ofwrinkles and streaks, and disappearance of the natural skin lines.

In order to prevent the above mentioned modifications and clinicalsymptoms, and to improve the appearance of the skin surface inparticular by reducing the wrinkle depth and eliminating fine wrinkles,it would be sensible to apply substances capable of simultaneouslyexerting the following effects:

-   -   activation of the synthesis reactions in the skin matrix by        stimulating the growth factor (TGFβ1) activity responsible for        the anabolism of the macromolecules of the extracellular matrix    -   reduction of the degradation reactions in the skin matrix by        modulating the metalloproteinase activity responsible for the        catabolism of the macromolecules in the extracellular matrix and        protection of these components from the influence of these        enzymes.

As collagen represents about 80% of the skin proteins, it is easilyunderstandable that the smallest diminution of its tissue concentrationmay have considerable consequences for the mechanical and physiologicalproperties of skin.

It has been surprisingly found that it is possible to synthesizecosmetically active tripeptides and derivatives thereof (hereinafterreferred to as “compounds of the present invention”) and topicallyapplicable, cosmetic compositions against chrono- and photo-induced skinaging (anti-aging products), which may diffuse rapidly and in sufficientconcentration through the cell membrane up to the intracellular site ofaction and produce a rapid and strong stimulation of collagen synthesis.This results from the capacity of the compounds of the present inventionto activate the synthesis reactions in the skin matrix by specificallystimulating the growth factor TGFβ1 responsible for the anabolism ofmacromolecules in the skin matrix.

Therefore, the compounds of the present invention exert a stimulatingeffect on the extracellular matrix, which decisively influences themechanical and physiological appearance of skin.

It could be shown that replacing the central amino acid in thetripeptide sequence RFK (Arg-Phe-Lys) of TSP-1 by an amino acid bearingan alkyl chain optionally substituted by hydroxy as a side chain,together with substituting the peptide with a penetration-enhancing,lipophilic group result in a quicker diffusion through the cell membraneup to the intracellular site of action in higher concentration and thatthe compounds of the present invention thus produce a quicker andstronger stimulation of collagen synthesis than the compoundelaidyl-Lys-Phe-Lys corresponding to the state-of-the-art and describedin the patent application FR 2810323 published on 21 Dec. 2001.

SUMMARY OF THE INVENTION

The present invention relates to tripeptides and tripeptide derivativesof general formula I

wherein

R¹ represents H, —C(O)—R⁶, —SO₂—R⁶ or —C(O)—XR⁶

R² and R⁴, independent of one another, represent (CH₂)_(n)—NH₂or(CH₂)₃—NHC(NH)NH₂,

n equals 1-4,

R³ represents linear or branched C₁-C₄-alkyl that is optionallysubstituted by hydroxy,

R⁵ and R⁶, independent of one another, represent hydrogen, optionallysubstituted (C₁-C₂₄)-alkyl, optionally substituted C₂-C₂₄-alkenyl,optionally substituted phenyl, optionally substituted phenyl-C₁-C₄-alkylor 9-fluorenylmethyl,

X represents oxygen (—O—) or —NH—; or

XR⁵ with X═O also represents the esters of α-tocopherol, tocotrienol orretinol,

as racemates or as pure enantiomers, as well as the salts thereof forapplication as cosmetic actives.

DETAILED DESCRIPTION OF THE INVENTION:

The above used, general terms are defined as follows: alkyl compriseslinear as well as branched alkyl groups. Examples thereof are methyl,ethyl, propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl,n-undecanyl, n-dodecanyl, n-tridecanyl, n-hexadecanyl, n-heptadecanyl,n-octadecanyl or n-nonadecanyl as unbranched residues and isopropyl,tert.butyl, isobutyl, sec.butyl, isoamyl as branched residues. R⁵ andR⁶, as optionally substituted alkyl and independent of one another,preferably represent (C₂-C₂₄)- alkyl, preferably (C₃-C₁₈)-alkyl.

Alkenyl has the denotation of a mono- or poly-unsaturated, optionallysubstituted alkyl group, such as e.g. 8(Z)-heptadecenyl,8(Z),11(Z)-heptadecadienyl, 4(Z),7(Z),10(Z),-13(Z)-nonadecatetraenyl,8(Z)-11-hydroxyoctadecenyl.

α-Tocopheryl means (D)-, (L)- or(DL)-2,5,7,8-tetramethyl-2-(4′,8′,12′-trimethyltridecyl)-6-chromanyl,tocotrienyl means any isomer of2,5,7,8-tetramethyl-2-(4′,8′,12′-trimethyl-3′,7′,11′-tridecatrienyl)-6-chromanyland retinyl means3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraen-1-yl.

The compounds of formula (I) together with acids can form mono- orpolyvalent, homogeneous or mixed salts, e.g. with inorganic acids, suchas hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; or with appropriate carboxylic acids, e.g. aliphatic mono- ordicarboxylic acids, such as formic acid, acetic acid, trifluoroaceticacid, trichloroacetic acid, propionic acid, glycolic acid, succinicacid, fumaric acid, malonic acid, maleic acid, oxalic acid, phthalicacid, citric acid, lactic acid or tartaric acid; or with aromaticcarboxylic acids, such as benzoic acid or salicylic acid; or witharomatic-aliphatic carboxylic acids, such as mandelic acid or cinnamicacid; or with heteroaromatic carboxylic acids, such as nicotinic acid;or with aliphatic or aromatic sulfonic acids, such as methanesulfonicacid or toluenesulfonic acid. Dermatologically tolerated salts arepreferred.

The general formula (I) includes all the possible isomeric forms as wellas mixtures thereof, e.g. racemic mixtures and mixtures of rotamers.

Compounds of general formula I, wherein

R¹ means —C(O)—R⁶,

R² and R⁴ mean (CH₂)_(n)—NH₂,

R³ means linear or branched C₁-C₄-alkyl,

R⁵ means hydrogen, (C₁-C₂₄)-alkyl, (C₂-C₂₄)-alkenyl, phenyl,phenyl-(C₁-C₄)-alkyl, preferably hydrogen, and

R⁶ means (C₁-C₂₄) -alkyl or C₂-C₂₄-alkenyl are preferred.

Furthermore, the following compounds are preferred:

Elaidoyl-Lys-Val-Lys-OH

Elaidoyl-Lys-Thr-Lys-OH

Palm-Lys-Thr-Lys-OH

Palm-Lys-Val-Lys-OH

Palm-Orn-Val-Lys-OH

Palm-Orn-Val-Dab-OH

Palm-Orn-Val-Dap-OH

Palm-Dab-Val-Lys-OH

Palm-Dab-Val-Dab-OH

Palm-Dab-Val-Dap-OH

Palm-Dap-Val-Lys-OH

Palm-Dap-Val-Dab-OH

Palm-Dap-Val-Dap-OH

Palm-Arg-Val-Lys-OH

Palm-Arg-Val-Dab-OH

Palm-Arg-Val-Dap-OH

Palm-Lys-Val-Lys-OH

Palm-Lys-Val-Orn-OH

Palm-Lys-Val-Dab-OH

Palm-Lys-Val-Dap-OH

Palm-Lys-Val-Arg-OH

Palm-Lys-Leu-Lys-OH

Palm-Lys-ILe-Lys-OH

Palm-Lys-ILe-Dab-OH

Palm-Lys-NVa-Dab-OH

Palm-Lys-tBuGly-Dab-OH

Palm-Lys-Leu-Dab-OH

Palm-Lys-ILe-Dap-OH

Palm-Lys-NVa-Dap-OH

Palm-Lys-tBuGly-Dap-OH

Palm-Lys-Leu-Dap-OH

Palm-Lys-NLe-Lys-OH

Palm-Lys-Ala-Lys-OH

Palm-Lys-Ser-Lys-OH

Palm-Lys-HSe-Lys-OH

Palm-Arg-Val-Arg-OH

Pentadecanoyl-Lys-Val-Dab-OH

Pentadecanoyl-Lys-Val-Dap-OH

Heptadecanoyl-Lys-Val-Dab-OH

Heptadecanoyl-Lys-Val-Dap-OH

Myristoyl-Lys-Val-Lys-OH

Myristoyl-Lys-Val-Dab-OH

Myristoyl-Lys-Val-Dap-OH

Lauroyl-Lys-Val-Lys-OH

Caprinoyl-Lys-Val-Lys-OH

Stearoyl-Lys-Val-Lys-OH

Stearoyl-Lys-Val-Dab-OH

Stearoyl-Lys-Val-Dap-OH

Oleolyl-Lys-Val-Lys-OH

Palm-Lys-Val-Dab-OMe

Palm-Lys-Val-Dab-OOctyl

Palm-Lys-Val-Dab-OCetyl

Palm-Lys-Val-Dab-NH₂

Palm-Lys-Val-Dab-NHBu

Palm-Lys-Val-Dab-NHOctyl

Palm-Lys-Val-Dab-NHCetyl

Palm-Lys-Val-Dap-OMe

Palm-Lys-Val-Dap-OOctyl

Palm-Lys-Val-Dap-OCetyl

Palm-Lys-Val-Dap-NH₂

Palm-Lys-Val-Dap-NHBu

Palm-Lys-Val-Dap-NHOctyl

Palm-Lys-Val-Dap-NHCetyl

C₁₄H₂₉—NH—CO-Lys-Val-Lys-OH

C₁₄H₂₉—NH—CO-Dab-Val-Dab-OH

C₁₄H₂₉—NH—CO-Lys-Ile-Dab-OH

C₁₄H₂₉—NH—CO-Lys-Val-Dap-OH

C₁₄H₂₉—NH—CO-Arg-Val-Arg-OH

C₁₄H₂₉—NH—CO-Dab-Val-Dap-OH

C₁₄H₂₉—NH—CO-Lys-Ile-Dap-OH

C₁₄H₂₉—NH—CO-Lys-Val-Dab-OH

C₁₄H₂₉—NH—CO-Dap-Val-Dap-OH

C₁₆H₃₃—NH—CO-Lys-Val-Lys-OH

C₁₈H₃₇—NH—CO-Lys-Val-Lys-OH

Boc-Lys-Val-Lys-OH

Ac-Lys-Val-Lys-OH

Z-Lys-Val-Lys-OH

Fmoc-Lys-Val-Lys-OH

C₈H₁₇—SO₂-Lys-Val-Lys-OH

C₁₆H₃₃—SO₂-Lys-Val-Lys-OH

H-Lys-Val-Lys-NH₂

H-Lys-Val-Lys-OH

H-Lys-Thr-Lys-OH

H-Lys-Val-Lys-OOctyl

H-Lys-Val-Lys-OCetyl

H-Lys-Val-Lys-ORetinyl

H-Lys-Val-Lys-OTocopheryl

H-Lys-Val-Lys-OBn

H-Lys-Val-Lys-NH-Cetyl

H-Lys-Val-Lys-NH-Octyl

H-Lys-Val-Lys-NH-Bn

The compounds of the present invention can be used in concentrationsranging between 0.5 and 5,000 ppm (w/w), preferably between 1 and 1000ppm (w/w), in the cosmetic end product. The compounds of the presentinvention can be used as a solution, a dispersion, an emulsion orencapsulated in carriers such as macro-, micro- or nanocapsules, inliposomes or chylomicrons, or enclosed in macro-, micro- ornanoparticles or in microsponges or absorbed on powdered organicpolymers, talc, bentonite and further inorganic carriers.

The compounds of the present invention can be used in any galenic form:O/W and W/O emulsions, milk, lotions, ointments, gelatinous and viscous,surfactant and emulsifying polymers, pomades, shampoos, soaps, gels,powders, sticks and pencils, sprays, body oils, face masks, plasters.

The compounds of the present invention as well as the cosmeticcompositions containing same are used for skin care products, inparticular against the formation and aggravation of wrinkles and againstall consequences of natural or accelerated (sun rays, pollution) skinaging.

The following compositions constitute a further aspect of the presentinvention:

The tripeptide derivatives of general formula (I) can be composed withat least one additional skin care active. These compositions may containadditional dermatologically acceptable carriers as well.

Additional skin care actives: In a preferred embodiment, where thecomposition should be in contact with human horny tissue, the additionalcomponents should be suitable for application to horny tissue, i.e.,when incorporated into the composition they are suitable for use incontact with human horny tissue without undue toxicity, incompatibility,instability, allergic response, and the like within the scope of soundmedical judgment. The CTFA Cosmetic Ingredient Handbook, Second Edition(1992) describes a wide variety of non-limiting cosmetic andpharmaceutical ingredients commonly used in the skin care industry,which are suitable for use in the compositions of the present invention.Examples of these ingredient classes include: abrasives, absorbents,aesthetic components such as fragrances, pigments/colorings/colorants,essential oils, skin sensates, astringents, etc. (e.g., clove oil,menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazeldistillate), anti-acne agents, anti-caking agents, antifoaming agents,antimicrobial agents (e.g., iodopropinyl butylcarbamate), antioxidants,binders, biological additives, buffering agents, bulking agents,chelating agents, chemical additives, colorants, cosmetic astringents,cosmetic biocides, denaturants, drug astringents, external analgesics,film formers or materials, e.g., polymers, for aiding the film-formingproperties and substantivity of the composition (e.g., copolymer ofeicosene and vinyl pyrrolidone), opacifying agents, pH adjusters,propellants, reducing agents, sequestrants, skin bleaching andlightening agents (e.g., alpha or beta arbutin, hydroquinone, kojicacid, ascorbic acid, magnesium ascorbyl phosphate, ascorbylglucosamine), skin-conditioning agents (e.g., humectants, includingmiscellaneous and occlusive), skin soothing and/or healing agents (e.g.,panthenol and derivatives (e.g., ethyl panthenol), aloe vera,pantothenic acid and its derivatives, allantoin, bisabolol, anddipotassium glycyrrhizinate), skin treating agents, thickeners, andvitamins and derivatives thereof.

In any embodiment of the present invention, however, the actives usefulherein can be categorized by the benefit they provide or by theirpostulated mode of action. However, it is to be understood that theactives useful herein can in some instances provide more than onebenefit or operate via more than one mode of action. Therefore,classifications herein are made for the sake of convenience and are notintended to limit the active to that particular application orapplications listed.

Farnesol: The topical compositions of the present invention may containa safe and effective amount of farnesol. Farnesol is a naturallyoccurring substance which is believed to act as a precursor and/orintermediate in the biosynthesis of squalene and sterols, especiallycholesterol. Farnesol is also involved in protein modification andregulation (e.g., farnesylation of proteins), and there is a cellnuclear receptor which is responsive to farnesol.

Chemically, farnesol is [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-1-oland as used herein “farnesol” includes isomers and tautomers of such.Farnesol is commercially available, e.g., under the names farnesol (amixture of isomers from Dragoco, 10 Gordon Drive, Totowa, N.J., USA) andtrans-trans-farnesol (Sigma Chemical Company, P.O. Box 14508, St. Louis,Mo., USA).

Phytantriol: The topical compositions of the present invention maycontain a safe and effective amount of phytantriol. Phytantriol is thecommon name for the chemical known as3,7,11,15-tetramethylhexadecane-1,2,3-triol. Phytantriol is useful,e.g., as a spider vessel/red blotchiness repair agent, a darkcircle/puffy eye repair agent, sallowness repair agent, a sagging repairagent, an anti-itch agent, a skin thickening agent, a pore reductionagent, an oil/shine reduction agent, a post-inflammatoryhyperpigmentation repair agent, a wound-treating agent, ananti-cellulite agent, and an agent for regulating skin texture,including wrinkles and fine lines.

Desquamation actives: A safe and effective amount of a desquamationactive may be added to the compositions of the present invention, morepreferably from about 0.1% to about 10%, even more preferably from about0.2% to about 5%, also preferably from about 0.5% to about 4%, by weightof the composition. Desquamation actives enhance the skin appearancebenefits of the present invention. For example, the desquamation activestend to improve the texture of the skin (e.g., smoothness). Onedesquamation system that is suitable for use herein contains sulfhydrylcompounds and zwitterionic surfactants and is described in U.S. Pat. No.5,681,852 to Bissett, cited herein by reference. Another desquamationsystem that is suitable for use herein contains salicylic acid andzwitterionic surfactants and is described in U.S. Pat. No. 5,652,228 toBissett, cited herein by reference. Zwitterionic surfactants such asdescribed in these applications are also useful as desquamatory agentsherein, with cetyl betaine being particularly preferred.

Anti-acne actives: The compositions of the present invention may containa safe and effective amount of one or more anti-acne actives. Examplesof useful anti-acne actives include resorcinol, sulfur, salicylic acid,benzoyl peroxide, erythromycin, zinc, etc. Further examples of suitableanti-acne actives are described in further detail in U.S. Pat. No.5,607,980, issued to McAtee on Mar. 4, 1997.

Anti-wrinkle actives/anti-atrophy actives: The compositions of thepresent invention may further contain a safe and effective amount of oneor more anti-wrinkle actives or anti-atrophy actives. Exemplaryanti-wrinkle/anti-atrophy actives suitable for use in the compositionsof the present invention include sulfur-containing D and L amino acidsand their derivatives and salts, particularly the N-acetyl derivatives,a preferred example of which is N-acetyl-L-cysteine; thiols, e.g.,ethane thiol; hydroxy acids (e.g., alpha-hydroxy acids such as lacticacid and glycolic acid or beta-hydroxy acids such as salicylic acid andsalicylic acid derivatives such as the octanoyl derivatives), phyticacid, lipoic acid; lysophosphatidic acid, skin peel agents (e.g., phenoland the like), vitamin B₃ compounds and retinoids which enhance thehorny tissue appearance benefits of the present invention, especially inregulating keratinous tissue condition, e.g., skin condition.

a) Vitamin B₃ compounds: The compositions of the present invention maycontain a safe and effective amount of a vitamin B₃ compound. Vitamin B₃compounds are particularly useful for regulating skin condition asdescribed in co-pending U.S. application Ser. No. 08/834,010, filed Apr.11, 1997 (corresponding to international publication WO 97/39733 A1,published Oct. 30, 1997). Exemplary derivatives of the foregoing vitaminB₃ compounds include nicotinic acid esters, including non-vasodilatingesters of nicotinic acid (e.g., tocopheryl nicotinate), nicotinyl aminoacids, nicotinyl alcohol esters of carboxylic acids, nicotinic acidN-oxide and niacinamide N-oxide.

b) Retinoids: The compositions of the present invention may also containa retinoid. As used herein, “retinoid” includes all natural and/orsynthetic analogs of vitamin A or retinol-like compounds which possessthe biological activity of vitamin A in the skin, as well as thegeometric isomers and stereoisomers of these compounds. The retinoid ispreferably retinol, retinol esters (e.g., C₂ to C₂₂ alkyl esters ofretinol, including retinyl palmitate, retinyl acetate, retinylpropionate), retinal, and/or retinoic acid (including all-trans retinoicacid and/or 13-cis-retinoic acid), more preferably retinoids other thanretinoic acid. Other suitable retinoids are tocopheryl retinoate[tocopherol ester of retinoic acid (trans- or cis-),adaptalene{6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid}, andtazarotene(ethyl6-[2-(4,4-dimethylthiochroman-6-yl)-ethinyl]nicotinate). Preferredretinoids are retinol, retinyl palmitate, retinyl acetate, retinylpropionate, retinal and combinations thereof. The compositions of thepresent invention may contain a safe and effective amount of theretinoid, such that the resultant composition is safe and effective forregulating horny tissue condition, preferably for regulating visibleand/or tactile discontinuities in skin, more preferably for regulatingsigns of skin aging, even more preferably for regulating visible and/ortactile discontinuities in skin texture associated with skin aging.

(c) Hydroxy acids: The compositions of the present invention may containa safe and effective amount of a hydroxy acid. Preferred hydroxy acidsfor use in the compositions of the present invention include salicylicacid and salicylic acid derivatives.

Peptides: Additional peptides, including but not limited to di-, tri-,tetra- and pentapeptides and derivatives thereof, may be included in thecompositions of the present invention in amounts that are safe andeffective. As used herein, “peptides” refers to both naturally occurringpeptides and synthesized peptides and also includes peptide mimetics andmetal complexes of “peptides”. Also useful herein are naturallyoccurring and commercially available compositions that contain peptides.

Suitable dipeptides for use herein include carnosine (β-Ala-His).Suitable tripeptides for use herein include Gly-His-Lys, Arg-Lys-Arg andHis-Gly-Gly. Preferred tripeptides and derivatives thereof includepalmitoyl-Gly-His-Lys, which may be purchased as Biopeptide CL™ (100 ppmof palmitoyl-Gly-His-Lys commercially available from Sederma, France);peptide CK (Arg-Lys-Arg); peptide CK+(Ac-Arg-Lys-Arg-NH₂); and a coppercomplex of Gly-His-Lys or of His-Gly-Gly sold as lamin from Sigma (St.Louis, Mo., USA). Suitable tetrapeptides for use herein include peptideE, Arg-Ser-Arg-Lys. Examples of pentapeptides are matrixyl(palmitoyl-Lys-Thr-Thr-Lys-Ser) available from Sederma, France, andthose described in WO 03/037933 (Pentapharm, Switzerland).

Antioxidants/radical scavengers: The compositions of the presentinvention may include a safe and effective amount of anantioxidant/radical scavenger. The antioxidant/radical scavenger isespecially useful for providing protection against UV radiation whichcan cause increased scaling or texture changes in the stratum corneumand against other environmental agents which can cause skin damage.

Antioxidants/radical scavengers such as ascorbic acid (vitamin C) andits salts, ascorbyl esters of fatty acids, ascorbic acid derivatives(e.g., magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbylsorbate), tocopherol (vitamin E), tocopherol sorbate, tocopherolacetate, other esters of tocopherol, butylated hydroxy benzoic acids andtheir salts, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(commercially available under the tradename Trolox™), gallic acid andits alkyl esters, especially propyl gallate, uric acid and its salts andalkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g.,N,N-diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g.,glutathione), dihydroxy fumaric acid and its salts, lycine pidolate,arginine pidolate, nordihydroguaiaretic acid, bioflavonoids, curcumin,lysine, 1-methionine, proline, superoxide dismutase, silymarin, teaextracts, grape skin/seed extracts, melanin, and rosemary extracts maybe used. Preferred antioxidants/radical scavengers are selected fromtocopherol sorbate and other esters of tocopherol, more preferablytocopherol sorbate. For example, the use of tocopherol sorbate intopical compositions applicable to the present invention is described inU.S. Pat. No. 4,847,071, issued on Jul. 11, 1989 to Donald L. Bissett,Rodney D. Bush and Ranjit Chatterjee.

Chelators: The compositions of the present invention may also contain asafe and effective amount of a chelator or chelating agent. As usedherein, “chelator” or “chelating agent” means an active agent capable ofremoving a metal ion from a system by forming a complex so that themetal ion cannot readily participate in or catalyze chemical reactions.The inclusion of a chelating agent is especially useful for providingprotection against UV radiation which can contribute to excessivescaling or skin texture changes and against other environmental agentswhich can cause skin damage.

Exemplary chelators that are useful herein are disclosed in U.S. Pat.No. 5,487,884, issued on Jan. 30, 1996 to Bissett et al., InternationalPublication No. 91/16035, Bush et al., published on Oct. 31, 1995 andInternational Publication No. 91/16034, Bush et al., published on Oct.31, 1995. Preferred chelators useful in the compositions of the presentinvention are furildioxime, furilmonoxime, and derivatives thereof.

Flavonoids: The compositions of the present invention may optionallycontain a flavonoid compound. Flavonoids are broadly disclosed in U.S.Pat. No. 5,686,082 and U.S. Pat. No. 5,686,367, both of which are hereinincorporated by reference. Flavonoids suitable for use in the presentinvention are flavanones selected from unsubstituted flavanones,mono-substituted flavanones, and mixtures thereof; chalcones selectedfrom unsubstituted chalcones, mono-substituted chalcones, di-substitutedchalcones, tri-substituted chalcones, and mixtures thereof; flavonesselected from unsubstituted flavones, mono-substituted flavones,di-substituted flavones, and mixtures thereof; one or more isoflavones;coumarins selected from unsubstituted coumarins, mono-substitutedcoumarins, di-substituted coumarins, and mixtures thereof; chromonesselected from unsubstituted chromones, mono-substituted chromones,di-substituted chromones, and mixtures thereof; one or more dicoumarols;one or more chromanones; one or more chromanols; isomers (e.g.,cis/trans-isomers) thereof and mixtures thereof. The term “substituted”as used herein means flavonoids, wherein one or more hydrogen atom hasbeen independently replaced with hydroxyl, C₁-C₈ alkyl, C₁-C₄ alkoxyl,O-glycoside, and the like or a mixture of these substituents.

Examples of suitable flavonoids include, but are not limited to,unsubstituted flavanone, mono-hydroxy flavanones (e.g., 2′-hydroxyflavanone, 6-hydroxy flavanone, 7-hydroxy flavanone, etc.), mono-alkoxyflavanones (e.g., 5-methoxy flavanone, 6-methoxy flavanone, 7-methoxyflavanone, 4′-methoxy flavanone, etc.), unsubstituted chalcone(especially unsubstituted trans-chalcone), mono-hydroxy chalcones (e.g.,2′-hydroxy chalcone, 4′-hydroxy chalcone, etc.), di-hydroxy chalcones(e.g., 2′,4-dihydroxy chalcone, 2′,4′-dihydroxy chalcone, 2,2′-dihydroxychalcone, 2′,3-dihydroxy chalcone, 2′,5′-dihydroxy chalcone, etc.), andtri-hydroxy chalcones (e.g., 2′,3′,4′-trihydroxy chalcone,4,2′,4′-trihydroxy chalcone, 2,2′,4′-trihydroxy chalcone, etc.),unsubstituted flavone, 7,2′-dihydroxy flavone, 3′,4′-dihydroxynaphthoflavone, 4′-hydroxy flavone, 5,6-benzoflavone, and7,8-benzoflavone, unsubstituted isoflavone, daidzein(7,4′-dihydroxyisoflavone), 5,7-dihydroxy-4′-methoxy isoflavone, soy isoflavones (amixture extracted from soy), unsubstituted coumarin, 4-hydroxycoumarin,7-hydroxy coumarin, 6-hydroxy-4-methyl coumarin, unsubstituted chromone,3-formyl chromone, 3-formyl-6-isopropyl chromone, unsubstituteddicoumarol, unsubstituted chromanone, unsubstituted chromanol, andmixtures thereof.

Preferred for use herein are unsubstituted flavanone, methoxyflavanones, unsubstituted chalcone, 2′,4-dihydroxy chalcone, andmixtures thereof. More preferred are unsubstituted flavanone,unsubstituted chalcone (especially the trans-isomer), and mixturesthereof.

Anti-inflammatory agents: The anti-inflammatory agent enhances the skinappearance benefits of the present invention, e.g., such agentscontribute to a more uniform and acceptable skin tone or color.

Steroidal anti-inflammatory agents, including but not limited to,corticosteroids such as hydrocortisone, hydroxyltriamcinolone,alpha-methyldexamethasone, dexamethasone phosphate, beclomethasonedipropionate, clobetasol valerate, desonide, desoxymethasone,desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasonediacetate, diflucortolone valerate, fluadrenolone, flucloroloneacetonide, fludrocortisone, flumethasone pivalate, fluosinoloneacetonide, fluocinonide, flucortine butylester, fluocortolone,fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide,hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone,triamcinolone acetonide, cortisone, cortodoxone, flucetonide,fludrocortisone, difluorosone diacetate, fluradrenolone,fludrocortisone, diflurosone diacetate, fluradrenolone acetonide,medrysone, amcinafel, amcinafide, betamethasone and the balance of itsesters, chloroprednisone, chlorprednisone acetate, clocortelone,clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide,fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate,hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate,triamcinolone, and mixtures thereof may be used. The preferred steroidalanti-inflammatory for use is hydrocortisone.

A second class of anti-inflammatory agents which is useful in thecompositions includes the non-steroidal anti-inflammatory agents. Thevariety of compounds encompassed by this group is well-known to thoseskilled in the art. For detailed disclosure of the chemical structure,synthesis, side effects, etc. of non-steroidal anti-inflammatory agents,one may refer to standard texts, including Anti-Inflammatory andAnti-Rheumatic Drugs, K. D. Rainsford, Vol. I-III, CRC Press, BocaRaton, (1985), and Anti-Inflammatory Agents, Chemistry and Pharmacology,1, R. A. Scherrer et al., Academic Press, New York (1974).

Specific non-steroidal anti-inflammatory agents useful in thecompositions of the present invention include, but are not limited to:

1) the oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam andCP-14,304;

2) the salicylates, such as aspirin, disalcid, benorylate, trilisate,safapryn, solprin, diflunisal and fendosal;

3) the acetic acid derivatives, such as diclofenac, fenclofenac,indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac,zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac,felbinac and ketorolac;

4) the fenamates, such as mefenamic, meclofenamic, flufenamic, niflumicand tolfenamic acids;

5) the propionic acid derivatives, such as ibuprofen, naproxen,benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen,indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,tioxaprofen, suprofen, alminoprofen and tiaprofenic acid; and

6) the pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone,azapropazone and trimethazone.

Mixtures of these non-steroidal anti-inflammatory agents may also beemployed as well as the dermatologically acceptable salts and esters ofthese agents. For example, etofenamate, a flufenamic acid derivative, isparticularly useful for topical application. Of the non-steroidalanti-inflammatory agents, ibuprofen, naproxen, flufenamic acid,etofenamate, aspirin, mefenamic acid, meclofenamic acid, piroxicam andfelbinac are preferred; ibuprofen, naproxen, ketoprofen, etofenamate,aspirin and flufenamic acid are more preferred.

Finally, so-called “natural” anti-inflammatory agents are useful in themethods of the present invention. Such agents may suitably be obtainedas an extract by suitable physical and/or chemical isolation fromnatural sources (e.g., plants, fungi, by-products of microorganisms) orcan be synthetically prepared. For example, candelilla wax, bisabolol(e.g., alpha bisabolol), aloe vera, plant sterols (e.g., phytosterol),Manjistha (extracted from plants of the genus Rubia, particularly RubiaCordifolia) and Guggal (extracted from plants of the genus Commiphora,particularly Commiphora Mukul), kola extract, chamomile, red cloverextract and sea alga extract may be used.

Additional anti-inflammatory agents useful herein include compounds ofthe liquorice (the plant genus/species Glycyrrhiza glabra) family,including glycyrrhetic acid, glycyrrhizic acid and derivatives thereof(e.g., salts and esters). Suitable salts of the foregoing compoundsinclude metal and ammonium salts.

Anti-cellulite agents: The compositions of the present invention mayalso contain a safe and effective amount of an anti-cellulite agent.Suitable agents may include, but are not limited to, xanthine compounds(e.g., caffeine, theophylline, theobromine and aminophylline).

Skin-tanning actives: Dihydroxyacetone, which is also known as DHA or1,3-dihydroxy-2-propanone, is a white to off-white, crystalline powder.The compound can exist as a mixture of monomers and dimers, with thedimers predominating in the solid crystalline state. Upon heating ormelting, the dimers break down to yield the monomers. This conversion ofthe dimeric form to the monomeric form also occurs in aqueous solution.One further example is erythrulose, available from Pentapharm,Switzerland. DHA and erythrulose can be administered in combination.

Skin-lightening agents: Suitable skin-lightening agents comprise thoseknown in the art, including kojic acid, arbutin, alpha-arbutin, ascorbicacid and derivatives thereof (e.g., magnesium ascorbyl phosphate orsodium ascorbyl phosphate) and extracts (e.g., mulberry extract,placental extract).

Skin-soothing and skin-healing actives: The compositions of the presentinvention may comprise a skin-soothing or skin-healing active.Skin-soothing or skin-healing actives suitable for use herein includepanthenoic acid derivatives (including panthenol, dexpanthenol, ethylpanthenol), aloe vera, allantoin, bisabolol and dipotassiumglycyrrhizinate.

Bisabolol: The topical compositions of the present invention may alsocontain a safe and effective amount of bisabolol. Bisabolol is anaturally occurring, unsaturated, monocyclic terpene alcohol having thefollowing structure:

It is the primary active component of chamomile extract/oil. Bisabololcan be synthetic (d,l-alpha-isomer or (+/−)-alpha-isomer) or natural((−)-alpha-isomer) in origin and can be used as essentially purecompounds or mixtures of compounds (e.g., extracts from natural sourcessuch as chamomile). The alpha form of bisabolol (a-bisabolol) is used ina variety of cosmetic products as a skin conditioning or soothing agent.As used herein, “bisabolol” includes chamomile extract or oil and anyisomers and tautomers of such. Suitable bisabolol compounds arecommercially available as a natural material from Dragoco (Totowa, N.J.,USA) under the product name alpha-bisabolol natural and as a syntheticmaterial from Fluka (Milwaukee, Wis., USA) under the product namealpha-bisabolol.

Antimicrobial and antifungal actives: The compositions of the presentinvention may contain an antimicrobial or antifungal active. Suchactives are capable of destroying microbes, preventing the developmentof microbes or preventing the pathogenic action of microbes. Preferredexamples of actives useful herein include those selected from salicylicacid, benzoyl peroxide, 3-hydroxy benzoic acid, glycolic acid, lacticacid, 4-hydroxy benzoic acid, acetyl salicylic acid, 2-hydroxybutanoicacid, 2-hydroxypentanoic acid, 2-hydroxyhexanoic acid, cis-retinoicacid, trans-retinoic acid, retinol, phytic acid, N-acetyl-L-cysteine,lipoic acid, azelaic acid, arachidonic acid, benzoylperoxide,tetracycline, ibuprofen, naproxen, hydrocortisone, acetominophen,resorcinol, phenoxyethanol, phenoxypropanol, 2,4,4′-trichloro-2′-hydroxydiphenyl ether, 3,4,4′-trichlorocarbanilide, octopirox, lidocainehydrochloride, clotrimazole, miconazole, ketoconazole, neocycin sulfateand mixtures thereof.

Sunscreen actives: Exposure to ultraviolet light can result in excessivescaling and texture changes of the stratum corneum. Therefore, thecompositions of the present invention may optionally contain a sunscreenactive. As used herein, “sunscreen active” includes both sunscreenagents and physical sunblocks. Suitable sunscreen actives may be organicor inorganic.

Inorganic sunscreens useful herein include the following metallicoxides: titanium dioxide, zinc oxide, zirconium oxide, iron oxide andmixtures thereof.

Examples of organic sunscreens are 2-ethylhexyl-p-methoxycinnamate(commercially available as PARSOL MCX), 4,4′-t-butylmethoxydibenzoyl-methane (commercially available as PARSOL 1789),2-hydroxy-4-methoxybenzophenone, octyldimethyl-p-aminobenzoic acid,digalloyltrioleate, 2,2-dihydroxy-4-methoxybenzophenone,ethyl-4-(bis(hydroxy-propyl))aminobenzoate,2-ethylhexyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexylsalicylate,glyceryl-p-aminobenzoate, 3,3,5-trimethylcyclohexylsalicylate,methylanthranilate, p-dimethylaminobenzoic acid or aminobenzoate,2-ethylhexyl-p-dimethylaminobenzoate, 2-phenylbenzimidazole-5-sulfonicacid, 2-(p-dimethylaminophenyl)-5-sulfonicbenzoxazoic acid, octocrylene,2,2′-methylene-bis-[6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)-phenol]available from Ciba SC as TINOSORB™ M,2,4-Bis-[(4-(2-ethylhexyloxy)-2-hydroxy)-phenyl]-6-(4-methoxyphenyl)-1,3,5-triazinavailable from Ciba SC as TINOSORB™ S and mixtures of these compounds.

Also particularly useful in the compositions are sunscreen actives suchas those disclosed in U.S. Pat. No. 4,937,370 issued to Sabatelli onJun. 26, 1990, and U.S. Pat. No. 4,999,186 issued to Sabatelli & Spirnakon Mar. 12, 1991. The sunscreen agents disclosed therein have, in asingle molecule, two distinct chromophore moieties which exhibitdifferent ultra-violet radiation absorption spectra. One of thechromophore moieties absorbs predominantly in the UVB radiation rangeand the other absorbs strongly in the UVA radiation range.

Preferred members of this class of sunscreens are4-N,N-(2-ethylhexyl)methyl-aminobenzoic acid ester of2,4-dihydroxybenzophenone; N,N-di-(2-ethylhexyl)-4-aminobenzoic acidester with 4-hydroxydibenzoylmethane;4-N,N-(2-ethylhexyl)methyl-aminobenzoic acid ester with4-hydroxydibenzoylmethane; 4-N,N-(2-ethylhexyl)methylaminobenzoic acidester of 2-hydroxy-4-(2-hydroxyethoxy)benzophenone;4-N,N-(2-ethylhexyl)methylaminobenzoic acid ester of4-(2-hydroxyethoxy)dibenzoylmethane;N,N-di-(2-ethylhexyl)-4-aminobenzoic acid ester of2-hydroxy-4-(2-hydroxyethoxy)benzophenone; andN,N-di-(2-ethylhexyl)-4-aminobenzoic acid ester of4-(2-hydroxyethoxy)dibenzoylmethane and mixtures thereof. Exact amountswill vary depending upon the sunscreen or sunscreens chosen and thedesired Sun Protection Factor (SPF).

Conditioning agents: The compositions of the present invention maycontain a conditioning agent selected from humectants, moisturizers orskin conditioners. These materials include, but are not limited to,guanidine, urea, glycolic acid and glycolate salts (e.g., ammonium andquaternary alkyl ammonium), salicylic acid; lactic acid and lactatesalts (e.g., ammonium and quaternary alkyl ammonium); aloe vera in anyof its variety of forms (e.g., aloe vera gel); polyhydroxy alcohols suchas sorbitol, mannitol, xylitol, erythritol, glycerol, hexanetriol,butanetriol, propylene glycol, butylene glycol, hexylene glycol and thelike; polyethylene glycols; sugars (e.g., melibiose) and starches, sugarand starch derivatives (e.g., alkoxylated glucose, fructose,glucosamine), hyaluronic acid, lactamide monoethanolamine, acetamidemonoethanolamine, panthenol, allantoin and mixtures thereof. Also usefulherein are the propoxylated glycerols described in U.S. Pat. No.4,976,953, to Orr et al., issued on Dec. 11, 1990.

Also useful are various C₁-C₃₀ monoesters and polyesters of sugars andrelated materials. These esters are derived from a sugar or polyolmoiety and one or more carboxylic acid moieties.

Preferably, the conditioning agent is selected from urea, guanidine,sucrose polyester, panthenol, dexpanthenol, allantoin and combinationsthereof.

Dermatologically acceptable carriers: The topical compositions of thepresent invention also contain a dermatologically acceptable carrier.The phrase “dermatologically acceptable carrier”, as used herein, meansthat the carrier is suitable for topical application to the hornytissue, has good aesthetic properties, is compatible with the actives ofthe present invention and any other components, and will not cause anyuntoward safety or toxicity concerns.

The carrier can be in a wide variety of forms. For example, emulsioncarriers, including, but not limited to, oil-in-water, water-in-oil,water-in-oil-in-water, and oil-in-water-in-silicone emulsions, areuseful herein.

A) Water-in-Silicone Emulsion

-   -   Water-in-silicone emulsions contain a continuous silicone phase        and a dispersed aqueous phase.

B) Oil-in-Water Emulsions

-   -   Other preferred topical carriers include oil-in-water emulsions        having a continuous aqueous phase and a hydrophobic,        water-insoluble phase (“oil phase”) dispersed therein. Examples        of suitable oil-in-water emulsion carriers are described in U.S.        Pat. No. 5,073,371 issued to D. J. Turner et al. on Dec. 17,        1991 and U.S. Pat. No. 5,073,372 issued to D. J. Turner et al.        on Dec. 17, 1991.

EXAMPLES

The following examples should illustrate the invention without limitingits scope. The following abbreviations are used in the text and inExamples 1-7:

AcOH: Acetic acid

AB: Antibody

Boc: tert.-Butyloxycarbonyl

BSA: Bovine serum albumine

Dab: 2,4-Diaminobutyric acid

Dap: 2,3-Diaminopropionic acid

DBU: 1,8-Diazabicyclo[5,4,0]undec-7-ene(1,5-5)

FCS: Foetal calf serum

TFA: Trifluoroacetic acid

Gly: Glycine

HSe: Homoserine

ILe: Isoleucine

MEM: Minimal essential medium

NEAA: Non essential amino acids

NLe: Norleucine

NVa: Norvaline

Orn: Ornithine

Palm: Palmitoyl

PBS: Phosphate buffered saline

Pe: Petroleum ether

RT: Room temperature

tBu: tert.-Butyl

tBuGly: tert.-Butylglycine

TBTU:O-(Benzotriazol-1-yl)-N,N,N′,N′,-tetramethyluronium-tetrafluoroborate

TGFβ1: Transforming growth factor-β1

Example 1 Determination of the Stimulation of Collagen Type I Synthesisin Fibroblast Cell Cultures by Treatment with the Tripeptide Derivativesof the Present Invention

Type I collagen of in-vitro cultured skin fibroblasts was detected withan ELISA (Enzyme-Linked Immunosorbent Assay). The increase in thecollagen production of the cells was quantified in the presence of thepeptidic actives by using this method.

Human skin fibroblasts were isolated from foreskin and bred in culturemedium.

After 72 h of incubation with the corresponding peptides (actives) thequantitative determination was performed using an antibody specific forcollagen I.

Material:

Culture Medium: Test medium: MEM MEM 10% FCS no FCS 100 IU/ml penicillin100 IU/ml penicillin 0.1 mg/ml streptomycin 0.1 mg/ml streptomycin 1 mMNEAA 1 mM NEAA 1 mM Na pyruvate 1 mM Na pyruvate 2 mM L-glutamine 2 mML-glutamine 20 mM HEPES buffer 20 mM HEPES buffer Washing buffer: Milksolution: 0.05M Tris, pH 8.5 washing buffer 0.15M NaCl 5% milk powder0.1% BSA 0.1% Tween-20 AB dilution solution: Substrate solution: 50 mlSuperblock (37515; 1 ImmunoPure ® OPD tablet Pierce) (34006; Pierce) 450ml H₂O 9 ml H₂O 0.05% Tween 1 ml stable peroxide substrate buffer, 10x(34062; Pierce)

The 1^(st) AB (MAB1340; Chemicon) and the 2^(nd) AB (31430; SocochimS.A.) are diluted 1/500 with AB dilution solution.

Method:

The fibroblasts are incubated until confluent at a density of approx.5000 cells per well in 96 well-plates in culture medium (37° C./5% CO₂)for 3 days. The medium is replaced with test medium with three differentconcentrations of test substance in triplicate. The following controlsare tested on each plate:

Negative controls: Positive controls: A) A) with cells with cellswithout 1^(st) AB; with 2^(nd) AB with 1^(st) and 2^(nd) AB B) B)without cells with cells with 1^(st) and 2^(nd) AB with 1^(st) and2^(nd) AB with 10 ng/ml TGF-β1 C) For each peptide a well without cellsis tested to exclude the unspecific binding of both AB.

After incubation of the plates for further 72 hours, the precipitatedcollagen I is detected and quantified according to the followingprotocol:

-   -   discard medium and wash with 200 μl/well of PBS    -   fix with 100 μl/well of methanol for 15 min at RT/shaker 600 rpm    -   discard methanol and block with 200 μl/well of milk solution for        30 min at RT/shaker 600 rpm    -   discard milk solution and incubate with 100 μl/well of the        1^(st) AB dilution for 2 h at RT/shaker 600 rpm    -   discard 1^(st) AB dilution and wash 3× with 200 μl/well of        washing buffer    -   incubate with 100 μl/well of the 2^(nd) AB dilution for 3 h at        RT/shaker 600 rpm    -   discard 2^(nd) AB dilution; wash 3× with 200 μl/well of washing        buffer and 1× with 100 μl/well of PBS    -   add 100 μl/well of substrate solution for 20 min at RT/shaker        600 rpm    -   stop the reaction with 50 μl/well of H₂SO₄ (2M) and measure at        492 nm.

TABLE 1 Collagen stimulation by ELISA: Conc./ % Stim- No Substance[□mol/L] ulation Control without active — 0 Reference compound A(Elaidoyl-Lys- 0.01/20.0 23 Phe-Lys-OH *2AcOH) 43 1Elaidoyl-Lys-Thr-Lys-OH *2AcOH 0.01 36 2 Elaidoyl-Lys-Val-Lys-OH *2AcOH0.01 30 3 Palm-Lys-Thr-Lys-OH *2AcOH 1.56 35 4 Palm-Lys-Val-Lys-OH*2AcOH 25.0 104 5 Palm-Dap-Val-Lys-OH *2TFA 50 35 6 Palm-Dap-Val-Lys-OH*2TFA 50 48 7 Myristoyl-Lys-Val-Lys-OH *2TFA 100 61 8Palm-Lys-Val-Orn-OH *2TFA 25 31 9 Palm-Lys-Ile-Lys-OH *2TFA 25 38 10H₂₉C₁₄—NH—CO-Lys-Val-Lys-OH *2TFA 50 63 11H₃₃C₁₆—NH—CO-Lys-Val-Lys-OH*2TFA 50 41 12H₃₇C₁₈—NH—CO-Lys-Val-Lys-OH*2TFA 50 25 13 Palm-Lys-Val-Dap-OH *2TFA 100154 14 Palm-Lys-Val-Dab-OH *2TFA 25 60 15 Palm-Arg-Val-Arg-OH *2TFA 5049

Example 2 Formulation of an Ointment

Method: Ingredients 1-5 (A) are heated to 70° C. Ingredients 6-7 (B) areheated to 75° C. Under stirring B is added to A, cooled to 50° C.,homogenized and cooled to 30° C. Afterwards, ingredients 8-9 (C) andingredient 10 (D) are added one after the other and the mixture isstirred cold.

No. Ingredient % w/w 1 (A) Tego Care 450 3.00 2 Cetearyl alcohol 2.25 3Glyceryl stearate 2.25 4 Cetiol 868 10.00 5 Squalane 5.00 6 (B)Deionized water 66.995 7 Sodium hyaluronate 5.00 8 (C) Glycerin 5.00 9Phenonip 0.5 10 (D) Palm-Lys-Val-Lys-OH 0.005

Example 3 Formulation of a Gel

Method: Ingredients 2-6 (A) are dissolved one after the other indeionized water. The pH is adjusted to 6.0 with ingredient 7 (B),whereupon ingredient 8 (C) is added.

No. Ingredient % w/w 1 (A) Deionized water 92.095 2 1,3-Butanediol 5.003 Phenonip 0.50 4 Abil B 8843 1.50 5 Carboxymethyl Cellulose 0.15 6Carbopol Ultrez 10 0.75 7 (B) NaOH 8 (C) Palm-Lys-Val-Lys-OH 0.005

Examples 4-8

The following embodiments 4-8 describe the synthesis of the compounds offormula (I) of the present invention and of salts of such compounds. Theeluates and products obtained according to the examples are analysedusing proton NMR, HPLC electrospray MS or microanalysis. The compoundscan be manufactured according to known methods described hereinafter(general instructions from M. Bodanszky “The Practice of PeptideSynthesis”, Springer, 2^(nd) Edition, 1994). Accordingly, the aminoacid, e.g. lysine, is bound to a resin at the carboxy terminus in asolid-phase synthesis, whereby its amino group is protected by aprotective group, e.g. by the Fmoc protective group. The side chain isprotected with, e.g., Boc or t-butyl. If necessary, the protectivegroups are selectively split off in order to link up the further aminoacid derivatives with the reagents commonly used in peptide synthesisuntil the desired chain is completely built up. Afterwards, the peptideis split off from the resin at the carboxy terminus and the crudepeptide is precipitated by instillation into an appropriate solventmixture. The mixture is purified by HPLC, optionally exchanged in theopposite ions and the substance is lyophilized.

Example 4 Elaidoyl-Lys-Thr-Lys-OH*2TFA

The protective peptide is built on 1.00 g (0.78 mmol) ofH-Lys(Boc)-2-chlorotrityl resin using Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OHand elaidoyl-OSu. The resin is treated for 30 min with 8 ml of TFA 95%and the solution is poured in drops into 100 ml of Et₂O. The precipitateis sucked off, washed, purified by preparative HPLC after drying andlyophilized.

Yield: 73 mg (0.097 mmol, 12%)

Example 5 H-Lys-Thr-Lys-OH*3TFA

The protective peptide is built on 3.00 g (2.58 mmol) ofH-Lys(Boc)-2-chlorotrityl resin using Fmoc-Thr(tBu)-OH and Z-Lys(Z)-OH.The resin is treated for 30 min with 20 ml of TFA 95% and the solutionis poured in drops into 400 ml of ^(t)BuOMe:PE=1:1. The precipitate issucked off, washed and purified by prep. HPLC after drying.

The partially protected peptide is dissolved in 50 ml ofdioxan:water=4:6, mixed with 200 mg Pd/C and 3 Eq TFA and reduced for 5h under H₂ atmosphere. The mixture is filtered through Celite, rotated,purified by prep. HPLC and lyophilized.

Example 6 Palm-Lys-Val-Lys-OH*2AcOH

The protective peptide is built on 1.00 g (0.80 mmol) ofH-Lys(Boc)-2-chlorotrityl resin using Fmoc-Val-OH, Fmoc-Lys(Boc)-OH andPalm-OSu. The resin is treated for 30 min with 8 ml of TFA 95% and thesolution is instilled in 100 ml of Et₂O. The precipitate is sucked off,washed and purified by prep. HPLC after drying. The substance is dilutedin 30 ml of dioxan:water=4:6, treated overnight with 2.0 g of BioRadresin (acetate form), filtered, rotated and lyophilized.

Yield: 110 mg (0.15 mmol, 19%)

Example 7 H-Lys-Val-Lys-NH-Cetyl*3TFA

The protective peptide is built on 13.5 g (10.8 mmol) ofH-Lys(Boc)-2-chlorotrityl resin using Fmoc-Val-OH and Boc-Lys(Boc)-OH.The resin is treated for 3*10 min with 80 ml of TFA 1% in methylenechloride and the solution is neutralized with pyridine:methanol solutionand purified by prep. HPLC.

Yield: Boc-Lys(Boc)-Val-Lys(Boc)-OH 4.66 g (6.915 mmol, 64%)

84 μl (0.441 mmol) of DIPEA, 78.5 mg (0.245 mmol) of TBTU and 59.1 mg(0.245 mmol) of cetyl amine are added to 150 mg (0.223 mmol) ofBoc-Lys(Boc)-Val-Lys(Boc)-OH in 5 ml of DMF. After 30 min the reactionsolution is submitted to an aqueous extraction and the residue from theorganic phase is treated for 30 min with TFA 95% and purified by prep.HPLC.

Yield: H-Lys-Val-Lys-NH-Cetyl*3TFA 48.2 mg (0.051 mmol, 23%)

Example 8 H-Lys-Val-Lys-O-Octyl*3TFA

10 ml of octanol are cooled to −10° C. and 75 μl of SOCl₂ (1.03 mmol)are carefully added. After 10 min 150 mg (0.223 mmol) ofBoc-Lys(Boc)-Val-Lys(Boc)-OH are added and the mixture is stirred for 3days. The product is obtained by purification over prep. HPLC.

Yield: H-Lys-Val-Lys-O-Octyl*3TFA 165.6 mg (0.200 mmol, 90%)

1-17. (canceled)
 18. A method for increasing the production of collagenand/or elastin in human skin and/or to delay or treat skin aging,thereby characterized that a compound of the general formula I

wherein R¹ represents —C(O)—R⁶, R² and R⁴ represent (CH₂)_(n)—NH₂, nequals 1-4, R³ represents linear or branched C₁-C₄ optionallysubstituted by hydroxy, R⁵ represents hydrogen, (C₁-C₂₄)-alkyl oroptionally substituted C₂-C₂₄-alkenyl, R⁶ represents (C₁-C₂₄)-alkyl oroptionally substituted C₂-C₂₄-alkenyl, and X represents oxygen (—O—) or—NH—; or XR⁵ with X═O also represents the esters of α-tocopherol,tocotrienol or retinol; with the s roviso that R¹ does not representβ-methoxyacryl, R² and R⁴ do not represent (CH₂)₄—NH₂, X does notrepresent oxygen and R⁵ does not represent methyl simultaneously or acosmetically active composition comprising at least one compound of theabove formula I is applied onto the skin.
 19. A method according toclaim 18 wherein said cosmetically active composition comprises at leastone compound of formula I in a quantity ranging between 0.5 ppm and1,000 ppm (w/w) calculated on the weight of the compound of formula Iand of the carrier(s).
 20. A method according to claim 19 wherein saidquantity is ranging between 1 ppm and 100 ppm (w/w).
 21. A methodaccording to claim 18 wherein the cosmetically active compositioncontains (a) at least one compound of formula I and (b) a safe andeffective quantity of at least one additional skin care active selectedfrom the group comprising desquamation actives, anti-acne agents,vitamin B3 compounds, retinoids, di-, tri-, tetra- and pentapeptides andderivatives thereof, hydroxy acids, radical scavengers,anti-inflammatory agents, skin-tanning actives, skin-whitening agents,anti-cellulite agents, flavonoids, antimicrobial actives, skin-healingagents, antifungal actives, sunscreens, farnesol, phytantriol,allantoin, glucosamine and mixtures thereof.
 22. A method according toclaim 21 thereby characterized in that said cosmetically activecomposition comprises a dermatologically acceptable carrier.
 23. Amethod according to claim 18 wherein said cosmetically activecomposition can be in the form of a solution, a dispersion, an emulsionor encapsulated in carriers.
 24. A method according to claim 23, whereinthe cosmetically active composition is in macro, micro- or nanocapsules,in liposomes or chylomicrons, or enclosed in macro-, micro- ornanoparticles or in microsponges or absorbed on powdered organicpolymers, talc, bentonite and other mineral carriers.
 25. A methodaccording to claim 23 wherein said cosmetically active composition canbe in the form of characterized that it can be in the form of anemulsion, a milk, a lotion, an ointment, a gel-forming and viscous,surfactant and emulsifying polymer, a pomade, a shampoo, a soap, a gel,a powder, a stick or pencil, a spray, a body oil, a face mask or aplaster for transdermal application.
 26. A method according to claim 18wherein said cosmetically active composition is useful for increasingthe production of collagen and elastin in human skin, as a skin careproduct, in particular to prevent the formation and aggravation ofwrinkles and against all consequences of natural (endogenous) oraccelerated (exogenous) skin aging.